Method and apparatus for detection of fluorescently labeled materials

ABSTRACT

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

BACKGROUND OF THE INVENTION

The invention provides a method and associated apparatus for detectingand analyzing reactions of fluorescently marked materials on a singlesubstrate surface.

Certain macromolecules are known to interact and bind to other moleculeshaving a very specific three-dimensional spatial and electronicdistribution. Any large molecule having such specificity can beconsidered a target. The various molecules that targets selectively bindto are known as probes.

Methods and devices for detecting fluorescently marked targets ondevices are known. Generally, the devices includes a microscope and amonochromatic or polychromatic light source for directing light at asubstrate. A photon counter detects fluorescence from the substrate,while an x-y translation stage varies the location of the substrate. Acomputer controls the movement of the x-y translation table and datacollection. Such devices are discussed in, for example, U.S. Pat No.5,143,854 (Pirrung et al.) incorporated herein by reference for allpurposes. See also PCT WO 92/10092 also incorporated herein by referencefor all purposes.

Light from the light source is focused at the substrate surface bymanually adjusting the microscope. Manual adjustment is, on occasion,time consuming and inconvenient. Moreover, due to inherent imperfectionspresent in the x-y translation table and substrate, there is apossibility that the substrate will be out of focus as it is moved fromone region to another. As a result, the data collected may bemisrepresented.

Also, temperature sometimes impact a chemical reaction between targetsand probes. Generally, targets are more active or form stronger bonds atlower temperatures while the converse is true at higher temperatures.However, if the temperature is too low, the binding affinity of thetarget may become excessively strong, thus causing target to bind withcomplements (matches) as well as non-compliments (mismatches). Hence,the ability to control temperature may affect optimum binding betweenthe targets and probes while minimizing mismatches.

In addition, the microscope detection devices are uneconomical to use.Typically, these devices incorporates the use of a microscope, and amultichannel scaler, both of which are costly.

From the above, it is apparent that an improved method and apparatus fordetecting fluorescently labeled targets on a substrate is desired.

SUMMARY OF THE INVENTION

Methods and devices for the detection of fluorescently labeled targetson a substrate are disclosed. The detection method and devices utilize asubstrate having a large variety of probes at known locations on itssurface. The substrate, when placed in a confocal detection device, isexposed to fluorescently labeled targets that bind to one or more of theprobes.

The confocal detection device includes a monochromatic or polychromaticlight source, means for directing an excitation light from the lightsource at the substrate, means for focusing the light on the substrate,means for controlling temperature of the substrate during a reaction,means for detecting fluorescence emitted by the targets in response tothe excitation light by directing the fluorescence through confocalpinholes, and means for identifying the region where the fluorescenceoriginated. The means for controlling the temperature may include atemperature controlled fluid filled flow cell. The means for detectingthe fluorescent emissions from the substrate, in some embodiments,include a photomultiplier tube. The means for focusing the excitationlight to a point on the substrate and determining the region thefluorescence originated from may include an x-y-z translation table.Further, translation of the x-y-z table, temperature control and datacollection are recorded and managed by an appropriately programmeddigital computer.

In connection with one aspect of the invention, methods for analyzingthe data collected by the fluorescent detection methods and devices aredisclosed. Data analysis includes the steps of determining fluorescentintensity as a function of substrate position from the data collected;removing "outliers" (data deviating from a predetermined statisticaldistribution); and calculating the relative binding affinity of thetargets from the remaining data. The resulting data are displayed as animage with the intensity in each region varying according to the bindingaffinity between targets and probes therein.

By using confocal optics, as well as focusing and temperature regulatingtechniques in conjunction with the data analysis methods, it is possibleto quickly and accurately determine the relationship between structureand activity of certain molecules. Therefore, the potential fordiscovering novel probes with desirable pattern of specificity forbiologically important targets is dramatically increased.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a detection system for locating fluorescent markers on thesubstrate;

FIG. 1b shows an alternative embodiment of a detection system forlocating fluorescent markers on the substrate;

FIG. 1c shows another embodiment of a detection system for locatingfluorescent markers on the substrate;

FIG. 2 is a flow chart illustrating the operation of the detectionsystem;

FIGS. 3a and 3b show another flow chart illustrating the focusing stepof the detection system;

FIGS. 4a and 4b show another flow chart illustrating the dataacquisition step of the detection system;

FIG. 4c shows the relationship among the counters in the dataacquisition board versus time.;

FIG. 5 is another flow chart illustrating the method of converting datarepresenting photon counts as a function of position to datarepresenting fluorescence intensity level as a function of position; and

FIGS. 6a and 6b show another flow chart illustrating the data analysisstep.

DESCRIPTION OF THE PREFERRED EMBODIMENT CONTENTS

I. Definitions

II. Details of One Embodiment of a Fluorescent Detection Device

III. Details of the Operation of a Fluorescent Detection Device

IV. Details of One Embodiment of Data Analysis to Determine RelativeBinding Strength of Targets

V. Conclusion

I. Definitions

The following terms are intended to have the following general meaningsas they are used herein:

1. Complementary: Refers to the topological compatibility or matchingtogether of interacting surfaces of a probe molecule and its target.Thus, the target and its probe can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other.

2. Probe: A probe is a molecule that is recognized by a particulartarget. Examples of probes that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies.

3. Target: A target is a molecule that has an affinity for a givenprobe. Targets may be naturally-occurring or manmade molecules. Also,they can be employed in their unaltered state or as aggregates withother species. Targets may be attached, covalently or noncovalently, toa binding member, either directly or via a specific binding substance.Examples of targets which can be employed by this invention include, butare not restricted to, antibodies, cell membrane receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants(such as on viruses, cells or other materials), drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. Targets are sometimesreferred to in the art as anti-probes. As the term targets is usedherein, no difference in meaning is intended. A "Probe Target Pair" isformed when two macromolecules have combined through molecularrecognition to form a complex.

II. Fluorescent Detection Device

FIG. 1a schematically illustrates a device used to detect fluorescentlylabeled targets on a substrate. Substrate 230 comprises a number ofpresynthesized probes on its surface 231. The substrate on which thesequences are formed may be composed from a wide range of material,either biological, nonbiological, organic, inorganic, or a combinationof any of these, existing as particles, strands, precipitates, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates, slides, etc. The substrate may have any convenient shape, suchas a disc, square, sphere, circle, etc. The substrate is preferably flatbut may take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whicha sample is located. The substrate and its surface preferably form arigid support on which the sample can be formed. The substrate and itssurface are also chosen to provide appropriate light-absorbingcharacteristics. For instance, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, or combinations thereof. Other substratematerials will be readily apparent to those of skilled in the art uponreview of this disclosure. In a preferred embodiment the substrate isflat glass or silica.

According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light. The surface may alsobe provided with reflective "mirror" structures for maximization ofemission collected therefrom.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or any of the above-listedsubstrate materials. In one embodiment, the surface will be opticallytransparent and will have surface Si--OH functionalities, such as thosefound on silica surfaces.

The array of probe sequences may be fabricated on the substrateaccording to the pioneering techniques disclosed in U.S. Pat. No.5,143,854 or PCT WO 92/10092, incorporated herein by reference for allpurposes. The combination of photolithographic and fabricationtechniques may, for example, enable each probe sequence ("feature") tooccupy a very small area ("site") on the support. In some embodiments,this feature site may be as small as a few microns or even a singlemolecule. For example, about 10⁵ to 10⁶ features may be fabricated in anarea of only 12.8 mm². Such probe arrays may be of the type known asVery Large Scale Immobilized Polymer Synthesis (VLSIPS™).

Substrate 230 is preferably transparent to a wide spectrum of light. Insome embodiments, substrate 230 is made of a conventional microscopeglass slide or cover slip. It is preferable that the substrate be asthin as possible while still providing adequate physical support.Preferably, the substrate is less than about 1 mm thick, more preferablyless than 0.5 mm thick. Typically the substrate is a microscope glassslide of about 0.7 mm or 700 μm thick. In alternative embodiments, thesubstrate may be made of quartz or silica.

Substrate 230 is mounted on a flow cell 220. Flow cell 220 is a bodyhaving a cavity 221 on a surface thereof. The cavity is between about 50and 1500 μm deep with a preferred depth of 1000 μm. The bottom of thecavity is preferably light absorbing so as to prevent reflection ofimpinging light. In addition, the flow cell may be impervious to light.

When mounted to the flow cell, the substrate seals the cavity except forinlet port 223 and outlet port 224. According to a specific embodiment,the substrate is mounted to the flow cell by vacuum pressure generatedfrom a vacuum pump 270. Optionally, one or more gaskets may be placedbetween the flow cell and substrate and the intervening space is held atvacuum to ensure mating of the substrate to the gaskets.

Reagents, such as fluorescein labeled targets (fluorescence peak atabout 530 nm) are injected into the cavity 221 through the inlet port223 by a pump 240 or by using a syringe. The pump may be, for example, aMasterflex peristaltic pump made by Cole-Parmer Instrument Co orequivalent. Within the cavity, the reagents bind with one or morecomplementary probes on surface 231 of the substrate. The reagents arecirculated into the cavity via inlet port 223 by the pump and exitthrough the outlet port 224 for recirculation or disposal.

Flow cell 220 permits the substrate to remain in constant contact withreagents during detection, thereby allowing the substrate to be inequilibrium with targets therein. This arrangement also permits the userto manipulate test conditions without dismounting the substrate. In someembodiments, the flow cell provides means for controlling thetemperature within the flow cell. The means for controlling temperaturemay be a recirculating bath device 260 that flows water through channelsformed in the flow cell. In the specific embodiment, device 260 is arefrigerated circulating bath with a Rb 232 interface, catalog number13270-615 distributed by VWR or equivalent. However, means such as acirculating air device, a resistance heater, a peltier device(thermoelectric cooler) or others may also be employed. Computer 190monitors and controls device 260, thereby maintaining the flow cell at adesired temperature. Computer 190 may be selected from a wide variety ofcomputers including, for example, a Gateway 486DX computer or a similarappropriately programmed computer.

Controlling the temperature in the flow cell is advantageous becausetemperature affects the chemical reaction between targets and probes.For example, the bond between the targets and probes is generallystronger at lower temperatures. However, if the temperature is too low,the binding affinity between targets and probes may become excessivelystrong so as to produce apparent (but erroneous) matches. Thus,temperature can be controlled to maximize the binding affinity ofcomplementary targets while minimizing mismatches.

Flow cell 220 is mounted on a x-y-z translation table 250. X representsthe horizontal direction; y represents the vertical direction; and zrepresents the direction into and away from the microscope objectivesuch that focusing may be performed. In some embodiments, the x-y-ztranslation table may be a Pacific Precision Laboratories ModelST-SL06R-B5M. Movement of the translation table is controlled bycomputer 190.

A light source 100 generates a beam of light to excite the fluoresceinlabeled targets in the flow cell. The light source may be a argon laserthat generates a beam having a wavelength of about 488 nm, which in someembodiments may be a model 2017 or model 161C manufactured bySpectra-Physics. Other lasers, such as diode lasers, helium neon lasers,dye lasers, titanium sapphire lasers, Nd:YAG lasers or others may alsobe employed. The laser is directed at surface 231 through an opticaltrain comprised of various optical elements which will be describedbelow in detail.

The beam generated by laser 100 is typically nearly collimated andnearly Gaussian. However, a spatial filter may be optionally located infront of laser 100 to improve the Gaussian profile of the beam. Thespatial filter may comprise of a lens 101, a confocal pinhole 103 and alens 102. Lens 101 and 102, for example, may be 1/2' diameter 50 mmfocal length anti-reflection coated plano convex glass lens orequivalent. Both lenses are configured such that both their back focalplanes coincide with confocal pinhole 103. Pinhole 103, for example, mayhave a aperture of 30 μm.

Thereafter, the light passes through a beam splitter 110 to a dichroicmirror 120. The beam splitter may be, for example, a non-polarizing 50%beam splitter cube made by Melles Griot model number 03BSC007 orequivalent while the dichroic mirror may be a LWP-45°S-488R/520T-1025made by CVI Laser Corp. or equivalent. The functions of the beamsplitter cube will later be described in more detail.

In some embodiments, dichroic mirror 120 passes light having awavelength greater than about 520 nm, but reflects light having awavelength of about 488 nm. Consequently, the 488 nm light from thelaser is reflected by dichroic mirror 120 toward optical lens 130.Optical lens 130, in the specific embodiment, is 1/2' diameter -50 mmfocal length anti-reflection coated plano-concave glass lens made byNewport or equivalent. The light then passes through a microscopeobjective 140 to substrate 230 for magnification of the image sample.Microscope objective 140, in some embodiments, may be a 10×0.3NAmicroscope objective, but other magnifications could also be used. In apreferred embodiment, the distance between lens 130 and microscopeobjective 140 is about 100 mm.

Microscope objective 140 focuses the light on surface 231, therebyexciting the fluorescein labeled targets. Preferably, the microscopeobjective produces a spot about 2 μm in diameter in its focal plane. Theoptical train described in the above embodiments produces a 2 μmdiameter focal spot when used with a laser which generates a beamdiameter of 1.4 mm, such as the Spectra-Physics model 2017.

In alternative embodiments, the 2 μm spot may be easily obtained whenother types of light sources with different beam diameters are used.Since the diameter of the focal spot is inversely proportional to thediameter of the collimated beam produced by lens 102, the desired spotsize may be achieved by varying the ratio of the focal lengths of thespatial filter. Alternatively, a beam expander may be used to expand orcompress the beam from the light source to obtain the desired spot size.For example, if the laser is a model 161C, which generates a beamdiameter of 0.7 mm, a 2 μm diameter focal spot may be achieved if theratio of the focal lengths of the lenses in the spatial filter is 1:2instead of 1:1. Thus, by varying the focal lengths of the lenses in thespatial filter and/or using a beam expander, the appropriate excitationspot size may be achieved from various beam diameters.

In a preferred embodiment, the laser power delivered to the sample 50μW. Depending on the light source used, a variable neutral densityfilter 310 may be inserted between the laser 100 and the optical trainto attenuate the power of the laser to the desired power level.

In response to the excitation light, fluorescein labeled targets in theflow cell fluoresce light having a wavelength greater than about 520 nm.The fluorescence will be collected by the microscope objective 140 andpassed to optical lens 130. Optical lens 130 collimates the fluorescenceand passes it to dichroic mirror 120. In practice, light collected bymicroscope objective contains both fluorescence emitted by thefluorescein and 488 nm laser light reflected from the surface 231.

The laser component reflected from the substrate is reflected bydichroic mirror 120 back to beam splitter 110. Beam splitter 110 directsthe laser component through a lens 175. The lens, in some embodiments,may be a 1/2' diameter 50 mm focal length anti-reflection coated planoconvex glass lens made by Newport, but equivalent thereof may be used.Lens 175 focuses the laser component to a photodiode 170. Preferably, aconfocal pinhole 171 is located between lens 175 and photodiode 170.Confocal pinhole transmits substantially only the reflected lightoriginating from the focal plane of the microscope to photodiode 170while reflected light originating from out-of-focus planes is blocked.In some embodiments confocal pinhole 171 has an aperture of 50 μm.Photodiode 170 generates a voltage corresponding to the intensity of thedetected light. Photodiode may be, for example, a 13 DSI007 made byMelles Griot or equivalent, or other light detection devices, such asphotomultiplier tube or avalanche photodiode may be used. Output fromthe detection device is used by computer 190 to focus the laser at apoint on surface 231 of substrate 230.

As for the fluorescent component, most of it will pass through thedichroic mirror 120 since its wavelength is greater than about 520 nm.The fluoresced light is then focused by a lens 125 to a photomultipliertube 160 for detecting the number of photons present therein. Lens 125,in a preferred embodiment, is a 1/2' diameter 50 mm focal lengthanti-reflection coated plano convex glass lens made by Newport, butequivalent lens may be used. A confocal pinhole 161 may be locatedadjacent to lens 125. Confocal pinhole transmits florescence originatingfrom the focal plane of the microscope objective and filters out lightoriginating from other planes, such as from the glass or reagent.Accordingly, the signal-to-noise ratio of the fluoresced light isincreased. Additionally, a filter 165 is preferably located betweenphotomultiplier tube and confocal pinhole 161. In a specific embodiment,the filter transmits light having a wavelength greater than about 515 nmsuch as an Omega Optical 515 EFLP. In an alternative embodiment, thefilter may transmit light having a wavelength between about 515 and 545nm such as a 530 DF30 made by Omega Optical. Thus, photomultiplier tube160 detects substantially only fluoresced light.

In the specific embodiment, photomultiplier tube 160 is a HamamatsuR4457P photomultiplier tube with Hamamatsu C3866preamplifier/discriminator. The Photomultiplier tube generatesapproximately a 2 mV pulse for each photon detected. Each of these 2 mVpulses is converted to a TTL pulse by the preamplifier/discriminator.The TTL pulses, each one corresponding to a photon detected by thephotomultiplier tube, are then collected by a data acquisition board210. The data acquisition board may be a National Instruments "Lab-PC+"or equivalent.

Data acquisition board 210, typically, contains an Intel 8254 orequivalent counter/timer chip. This chip contains three counters,counter 0, counter 1 and counter 2. Counter 0 controls the operations ofcounters 1 and 2 for collecting data. Preferably, counter 0 isprogrammed to generate a square wave with a period which is equal totwice the data acquisition time per pixel. The output of counter 0 iscoupled to an external circuit board 200 which provides logic forinverting the square wave. In a preferred embodiment, the invertedoutput of counter 0 is connected to the gate input of counter 2 whilethe non-inverted output is connected to the gate input of counter 1.

In a preferred embodiment, the data acquisition board is not be able toread or store the fast 10 ns pulses generated bypreamplifier/discriminator (it is too fast for the 8254 chip). To solvethis problem, external circuit board 200 may additionally provide meansfor slowing down the pulses. For example, the logic in external circuitboard 200 may convert these pulses to 50 ns pulses with at least a 50 nsinterval between pulses.

The output of the C3866 preamplifier/discriminator, via external circuitboard 200, is connected to the clock inputs of counters 1 and 2. Whencounter 1 or counter 2 is gated on, it counts pulses generated by thepreamplifier/discriminator; when it is gated off, it ceases to count andcomputer 190 reads the accumulated number of counts therein. After thecomputer reads the count from either counter 1 or 2, the counter isre-initialized on the first clock pulse after its gate input goes high.The initialization pulse is about a 50 ns pulse that is generated by thelogic in the external circuit board 200 about 50 ns after eachtransition of the square wave signal from counter 0. The data stored incounter 1 or 2 represents the photon count as a function of substrateposition.

After data are collected from a region of the substrate, substrate 230is moved so that light can be directed at a different region on thesubstrate. The process is repeated until all regions on the substratehave been scanned. Generally, regions that contain a complementary probewill tend to exhibit a higher photon count than regions that do notcontain a complementary probe.

Although the above embodiments have been described for use in detectingemissions of fluorescein excited by an 488 nm argon laser, it will beapparent to those skill in art that other dyes and excitation sourcesmay used by simply modifying the elements in the optical train. Forexample, dichroic mirror 120 may be changed accordingly to pass lighthaving a wavelength comparable to the fluorescence peak of the dye used,but reflect light from the excitation source. Also, filter 165 ischanged to pass substantially only light having a wavelength similar tothe fluorescence peak of the dye used. In this manner, the detectiondevice can be easily modified to accommodate other types of excitationlight and/or dyes.

FIG. 1b illustrates an alternative embodiment of the fluorescencedetection device shown in FIG. 1a. FIG. 1b is similar to the one shownin FIG. 1a and the common elements have been numbered with the samereference numerals. The main difference between this embodiment and thatof FIG. 1a is that a photodiode 180 is provided to detect a component ofthe light generated by laser 100. Light generated by the laser, as inFIG. 1a, is directed at the beam splitter. However, a component of thislight is directed to photodiode 180. Photodiode 180 generates a voltagevalue which is proportional to the laser power. This voltage signal isused by the computer 190 to monitor and control the laser power.

FIG. 1c illustrates an alternative embodiment of the fluorescencedetection device. FIG. 1c is similar to the embodiment shown in FIG. 1aand the common elements have been numbered with the same referencenumerals. However, the embodiment in FIG. 1c provides means fordetecting a second fluorescent color. Two color detection is requiredwhen two different types of targets, each labeled with a different dye,are exposed to a substrate synthesized with probes. In some embodiments,fluorescein and rhodamine dyes may be used to label two different typesof targets respectively. Typically, each dye will have a fluorescencepeak at different wavelengths. For example, the fluorescence peak offluorescein is about 530 nm while that of a typical rhodamine dye isabout 580 nm.

To detect the second fluorescent color, a second dichroic mirror 300 isemployed. If rhodamine and fluorescein were used, then dichroic mirror300 is designed to pass light having a wavelength greater than about 570nm (rhodamine emissions) and reflect light having a wavelength less thanabout 560 nm (fluorescein emissions). Light with a wavelength less than560 nm is reflected to a lens 126 and through a confocal pinhole 151.Lens 126, may be equivalent to lens 125 while confocal pinhole 151 maybe similar to confocal pinhole 161. Filter 155 transmit the light to asecond photomultiplier tube 150. Filter 155 may be an Omega Optical530DF30 or equivalent that passes light with a wavelength between about515-545 nm. This ensures that substantially only fluorescein emissionsare detected by the photomultiplier 150.

On the other hand, light having a wavelength greater than 570 nm passesthrough dichroic mirror 300 to a lens 125. Lens 125 then directs thelight through pinhole 161 and filter 165 to photomultiplier tube 160.Filter 165 may be a Schott OG570 or equivalent which passes light havinga wavelength greater than 570 nm, thereby ensuring substantially onlyrhodamine emissions are detected by photomultiplier 160.

Output of the preamplifier/discriminator from the photomultiplier tube150 is processed by the external circuit board 200 before beingconnected to counters 1b and 2b on the data acquisition board 205. Datacollection by counters 1b and 2b are controlled by counter 0 from dataacquisition board 210 which generates a square wave to the gated inputsof the counters 1 and 2 via the external circuit board 200, similar tothat of counters 1 and 2 on data acquisition board 210.

According to the embodiment in FIG. 1c, two fluorescence colors can bedetected by employing a second dichroic mirror, photomultiplier tube andassociated lens, confocal pinhole and filter. The embodiment illustratedin FIG. 1c may be expanded by one skilled in the art to detect more thantwo fluorescence colors by employing an additional dichroic mirror,photomultiplier tube and associated lens, confocal pinhole and filterfor each additional fluorescence color to be detected.

III. Details on the Operation of a Fluorescent Detection Device

In the specific embodiment, data are acquired continuously along a linewhich is broken up into data points or pixels. The pixel size preferablyranges from 1 to 100 μm. Since pixels are preferably square, thedistance between scan lines will generally be between 1 to 100 μm. Eachof these pixels will be used to form a digital image. The imageresolution (i.e., the degree of discernable detail) depends upon thenumber of pixels that forms the image. The greater the number of pixels,the closer the digitized data will approximate the original image.Therefore, it is desirable to have as many pixels as possible. However,the number of pixels is directly related to the length of time requiredto scan the sample. In practice, it is found that having about 16 to 100pixels per feature (or about 4-10 pixels along the length of thefeature) is preferable to achieve the desire resolution in a reasonableamount of time.

The number of photons that is detected in each pixel is contingent uponseveral factors. The most obvious factor, of course, is the amount offluorescently labeled targets present in the pixel. Other, but not soobvious, factors include the intensity of the excitation light, lengthof time that the targets are excited, and quantum efficiency of thephotocathode at the fluorescence emission wavelength.

For example, exciting a region of about 2 μm with 50 μW of power willyield approximately 1600 W/cm² or 3.9×10²¹ photons/(sec cm²). At thisintensity, the fluorescence rate is Q_(e) k_(a) k_(f) /(k_(a)+k_(f))=1.1×10⁶ /sec (see Table I) with a photodestruction rate of Q_(b)k_(a) k_(f) /(k_(a) +k_(f))=36/sec. In a typical substrate synthesizedwith polymer sequences at about 5 nm apart, approximately 4×10⁴molecules/μm² or 1.25×10⁵ molecules will be present in the excitationvolume. If it is estimated that about 1% of these sequences bind withfluorescein labeled targets, then about 1250 molecules are excited or1.4×10⁹ fluorescence photons are generated per second. However, in atypical detection device, only about 2% of these photons are collectedby the microscope objective while about 98% never even make it into theoptical train of the detection device. Of the 2% collected, about 50%are lost in the optical train and of the remaining photons, only about10% are detected by the photomultiplier tube due to quantum efficiencyof the photocathode at the fluorescein emission wavelength. Thus,approximately 1.4×10⁶ photons might be counted per second. From theabove, it is apparent that these factors affect the number of photonsdetected at each pixel.

                  TABLE I                                                         ______________________________________                                        Fluorescein Optical Parameters                                                [Tsien, R. Y., Waggoner, A. Fluorophores for confocal                         microscopy: photophysics and photochemistry. In                               Handbook of Biological Confocal Microscopy; Pawley, J.,                       Ed.; Plenum Press: New York, 1990; pp. 169-178]                               ______________________________________                                        Absorption cross section, σ,                                                                    3.06 × 10.sup.-16                               cm.sup.2 molecules.sup.-1                                                     Fluorescence rate constant, k.sub.f, s.sup.-1                                                         2.2 × 10.sup.8                                  Absorption rate constant (1600 W/cm.sup.2)                                                            1.2 × 10.sup.6                                  k.sub.a, s.sup.-1                                                             Fluorescence quantum efficiency, Q.sub.c                                                              0.9                                                   Photodestruction efficiency, Q.sub.b                                                                  3 × 10.sup.-5                                   ______________________________________                                    

In the present invention, it is preferable to detect as many photons aspossible, preferably about 1000 photons per pixel in pixels containingcomplementary probes and targets because the signal-to-noise ratio isequal to the square root of the number of photon counts. Since thenumber of photons collected will vary according to the power and lengthof time the targets are excited by the light, the signal-to-noise ratiomay be improved by increasing the laser power or length of time thesubstrate is exposed to the laser or a combination thereof.

FIGS. 2-6 are flow charts describing a specific embodiment. FIG. 2 is anoverall description of the system's operation. Referring to FIG. 2, thedetection system is initialized at step 200. At step 201, the systemprompts the user for test parameters such as:

a) temperature of the substrate;

b) number of scans to be performed;

c) time between scans;

d) refocus between scans;

e) pixel size;

f) scan area; and

g) scan speed.

The temperature parameter controls the temperature at which detection isperformed. Temperature may vary depending on the type of polymers beingtested. Preferably, testing is done at a temperature that producesmaximum binding affinity while minimizing mismatches.

The "number of scan" parameter corresponds to the number of times theuser wishes to scan the substrate while the "time between scans"parameter controls the amount of time to wait before commencing asubsequent scan. In this manner, the user may perform a series of scansand if desired, each at a different temperature. Preferably, the timebetween scans is chosen to allow the system to reach chemicalequilibrium before commencing a subsequent scan.

In an alternative embodiment, means may be provided to increase thetemperature at set increments automatically after each scan. Further,the user may optionally choose to refocus the substrate after each scan.

The pixel size parameter dictates the size of each data collectionpoint. Generally, the size is chosen which results in at least 16 datacollection points or pixels per synthesis region ("feature").

Scan area parameter corresponds to the size of the substrate to betested. Scan speed parameter sets the length of time the laser exciteseach pixel. The slower the speed, the higher the excitation energy perpixel which will result in higher fluorescence count per pixel. Thus,increasing the laser power or decreasing the scan speed or a combinationthereof will increase the photon count in each pixel. Typically, thescan rate is set to generate approximately a count of 1000 photons forpixels having fluorescently-marked targets.

At step 202, the system initializes the x-y-z translation table bylocating the x-y-z stages at their home position. At step 203, thesystem focuses the laser on the surface 231 of the substrate. At step204, the system locates the x-y-z table at its start position. At step205, the system begins to translate the vertical stage, therebycollecting a series of data points over a vertical line at step 206.When a line of pixels has been scanned at step 207, the x-y-ztranslation table moves the horizontal stage to collect data from thenext line of pixels at step 208. The collected data is written to thefile as the substrate is repositioned at the top of the next line. Steps205 through 208 are repeated until data from all regions have beencollected. At step 209, the system determines if there are any morescans to be performed according to the set up parameters. If there are,the system at steps 210 and 211 determines the amount of time to waitbefore commencing the next scan and to either repeat the process fromstep 203 (if refocusing of the substrate is desired) or 204. Otherwise,the scan is terminated.

FIGS. 3a and 3b illustrate the focusing step 203 in greater detail.Auto-focusing is accomplished by the system in three phases. In thefirst phase, the system focuses the laser roughly on the back surface ofthe substrate. At step 301, the laser light is directed at one corner ofthe substrate, The laser light reflected by the substrate passes throughthrough microscope objective 140 and optical lens 130 to dichroic mirror120. The dichroic mirror reflects the light to beam splitter 110 whichguides the beam to lens 175. Lens 175 focuses the light through apinhole 171 to photodiode 170. Pinhole 171 is a confocal pinhole, whichtransmits light that is reflected from the surface located at the focalplane of the microscope objective and blocks light that is reflected orscattered from other surfaces. At step 302, the computer reads thephoto-diode's voltage, which is proportional to the amount of reflectedlight transmitted through pinhole 171. The flow cell is then moved about10 microns closer to the microscope objective at step 315, and theprocess from step 302 is repeated. The loop commencing at step 302 isrepeated until the voltage generated by the photodiode has peaked (i.e.,the present voltage value is less than the previous voltage value), atwhich time, the laser is roughly focused on the backside of thesubstrate. Since 10 μm steps are taken, the light is focused slightlyinside the substrate.

At step 304, the system continues with the next focusing phase by movingthe flow cell closer to the microscope objective. In a preferredembodiment, the distance over which the flow cell is moved is aboutequal to half the thickness of the substrate. The default distance is350 mm (1/2 the thickness of a typical substrate used) or a distanceentered by the user representing half the thickness of the substrateused. Again, at step 305, photo-diode 170 generates a voltagecorresponding to the intensity of the reflected light. Preferably, theflow cell is then moved about 10 microns closer at step 316, and theprocess from step 305 is repeated. The loop commencing at step 305 isrepeated until the voltage generated by the photodiode has peaked, atwhich time, the laser is roughly focused on a point beyond surface 231.

At step 307, the flow cell is moved farther from the microscopeobjective in steps of about 1 μm. The computer reads and stores thevoltage generated by the photodiode at step 308. At step 309, theencoder indicating the position of the focus stage is read and theresulting data is stored. The encoder determines the location of thefocus stage to within about 1 micron. At step 310, the system determinesif the present voltage value is greater then the previous value. If itis, the flow cell is then moved about 1 micron farther at step 317. Dueto the presence of noise, the process from step 308 is repeated evenafter the voltage value has peaked. Specifically, the process iscontinued until the voltage generated by the photodiode 104 is less thanthe peak voltage minus twice the typical peak-to-peak photodiode voltagenoise. At step 311, the data points are fitted into a parabola wherex=encoder position and y=voltage corresponding to the position. Fittingthe data points to a parabola gives a more accurate method of focusingthan by merely taking the highest point due to the presence of noise inthe data. At step 312, the system determines the focus position onsurface 231 which corresponds to the maximum of the parabola.

At step 313, the system determines whether all four corners have beenfocused. If they have been, then the process proceeds to step 314.Otherwise, the system proceeds to step 318 wherein it positions thesample by moving the x-y-z translation stage to focus the next corner.Thereafter, the system repeats the focusing process beginning at step301. The focussing process beginning at step 301 is repeated until theall four corners of the substrate has been focused.

At step 314, the system assumes that the substrate is planar. Thus, thefocus position of other pixels are obtained by interpolating the valuescollected from the focusing process. In this manner, auto-focusing foreach pixel of the substrate is achieved.

By using the focusing method disclosed herein, the laser is focused onsurface 231 of the substrate, which is significantly less reflectivethan the backside of the substrate. Generally, it is difficult to focuson a weakly reflective surface in the vicinity of a strongly reflectivesurface. However, this problem is solved by the present invention andthus, more accurate reading of the fluorescently marked targets isachieved.

FIGS. 4a and 4b illustrate the data acquisition process beginning atstep 205 in greater detail. In a specific embodiment, data are collectedby repeatedly scanning the substrate in vertical lines until the sampleis completely scanned. However, other techniques such as repeatedlyscanning the substrate in horizontal lines, bidirectional scanning(acquiring data in both directions) or others may be employed.

At step 401, the x-y-z translation table is initialized at the startposition. At step 402, the system calculates the constant speed at whichthe vertical stage is to be moved. The speed of the vertical stage isdetermined from the scan speed information entered by the user.Typically the scan speed is about 10 to 30 mm/sec or a speed at which aphoton count of about 1000 photons will be generated for regions havingcomplementary probes. At step 403, the system calculates the constantspeed at which the focusing stage is to be moved in order to maintainthe substrate surface 231 in focus. This speed is derived from the dataobtained during the focusing phase.

At step 404, the system calculates the number of pixels per line bydividing the length of the scan line by the pixel size. At step 405, thesystem initializes counter 0 on the data acquisition board with a valuesuch that a square wave having a period that is equal to twice the dataacquisition time per pixel is generated. The period is calculated bydividing the pixel size information entered by the user at step 209 bythe speed of the vertical stage derived from step 402. Counter 0 countsdown until it reaches zero, at which time, a square wave transitionoccurs (i.e., value of the square wave goes from low to high or high tolow), which causes counter 0 to be re-initialized.

In a preferred embodiment, counter 1 on the data acquisition board isconfigured to store the photon counts from the even pixels while counter2 is configured to store counts from the odd pixels, but otherconfigurations may be employed. At step 406, counter 2 is initialized tozero by the rising edge of the first period of the square wave.Thereafter, counter 2 is enabled and begins to collect data at step 407.

The system, at step 408, polls counter 0 and compares the present valueof counter 0 with its previous value. If the present value is less thanthe previous value, then counter 2 continues to accumulate photoncounts. On the other hand, if the present value in counter 0 is greaterthan its previous value, a square wave transition has occurred. Thefalling edge of the square wave disables counter 2 from counting, thus,completing the scan of the first pixel. Simultaneously, counter 1 isinitialized because its gate input is coupled to the inverted output ofcounter 0. The operation of counter 1 will be described in more detailduring the discussion on the second pass of the loop beginning at step406.

While counter 2 is disabled, the photon count stored in counter 2 isread at step 409. At step 410, the data is written and stored in memory,for example, in the form of a data structure, an array, a table or otherlisting means. In the alternative, the data may also be written to adata file. At step 411, the system determines if there are more pixelsin the line to scan. If there are, process repeats the steps beginningat 406. Otherwise, the system proceeds to step 412.

On the second pass of the loop beginning at step 405, the invertedfalling edge (rising edge) of the square wave initializes and enablescounter 1 to collect data at steps 406 and 407 respectively. At step408, the inverted rising edge (falling edge) of the square wave disablescounter 1 and data therein is read at step 409 and written to thecomputer at step 410.

In the specific embodiment, the counters are preferably configured tocollect and store data alternately, i.e., when counter 1 collects data,counter 2 stores data. FIG. 4c illustrates the relationship among thecount value in counter 0, the square wave, counter 1 and counter 2versus time.

At step 411, the system determines if there are more pixels left toscan. The loop from step 405 through step 411 is repeated until allpixels in the line have been scanned. After each line has been scanned,the system at step 412 calculates a gray scale for imaging. In apreferred embodiment, the gray scale contains 16 gray levels, but ascale of 64, 256 or other gray levels may be used. In alternativeembodiments, a color scale may be used instead of a gray scale.Preferably, the middle of the scale corresponds to the average countvalue detected during the scan. Thereafter, the raw data points arestored in a data file at step 413. At step 414, the scaled datarepresenting an image of the scanned substrate regions may be displayedon a screen or video display mean in varying shades of gray or colors.Each shade or color corresponds to the intensity level of fluorescenceat the respective regions.

While the image of the previous scanned line is being displayed, thesystem determines if there are any more lines to scan at step 415. Ifso, the horizontal stage is translated in preparation for scanning thenext line at step 416. The distance over which the horizontal stage ismoved is equal to about 1 pixel. Simultaneously, the vertical stage ismoved to the top of the next scan line. Thereafter, the system repeatsthe process starting at step 405 for the next scan line. The loop fromstep 405 to step 415 is repeated until scanning of the substrate iscompleted. In this manner, the system simultaneously displays andcollects data. Upon completion, the system creates a data file whereinthe data represents an array of photon counts as a function of substrateposition.

By counting the number of photons generated in a given area in responseto the excitation light, it is possible to determine where fluorescentlymarked molecules are located on the substrate. Consequently, it ispossible to determine which of the probes within a matrix of probes iscomplementary to a fluorescently marked target.

According to preferred embodiments, the intensity and duration of thelight applied to the substrate is controlled by the computer accordingto the set up parameters entered at step 201. By varying the laser powerand scan stage rate, the signal-to-noise ratio may be improved bymaximizing fluorescence emissions. As a result, the present inventioncan detect the presence or absence of a target on a probe as well asdetermine the relative binding affinity of targets to a variety ofsequences.

In practice it is found that a target will bind to several probesequences in an array, but will bind much more strongly to somesequences than others. Strong binding affinity will be evidenced hereinby a strong fluorescence signal since many target molecules will bind tothat probe. Conversely, a weak binding affinity will be evidenced by aweak fluorescence signal due to the relatively small number of targetmolecules which bind in a particular probe. As such, it becomes possibleto determine relative binding avidity (or affinity in the case ofunivalent interactions) of a probe herein as indicated by the intensityof a fluorescent signal in a region containing that probe.

Semiquantitative data on affinities may also be obtained by varying setup conditions and concentration of the targets in the reagent. This maybe done by comparing the results to those of known probe/target pairs.

While the detection apparatus has been illustrated primarily herein withregard to the detection of marked targets, the invention will findapplication in other areas. For example, the detection apparatusdisclosed herein could be used in the fields of catalysis, DNA orprotein gel scanning, and the like.

IV. Data Analysis System to Determine Relative Binding Strength ofTargets

Before the data file representing an array of photon counts as afunction of position is analyzed to determine the relative bindingaffinity of targets, the data file is preferably converted to an imagefile wherein the data is indicative of fluorescence intensity level as afunction of substrate position.

FIG. 5 illustrates the process for converting or scaling the data fromphoton counts to fluorescence intensity level in greater detail. Theconversion procedure is started by prompting the operator for the nameof data file of interest. At step 501, the system retrieves thespecified data file for analysis.

At step 502, the user directs the system to scale the data either inrelative range or absolute range mode. In the absolute range mode, thedata is scaled by using the set of values between the minimum andmaximum number of photons. If absolute range mode is chosen, the systemproceeds to step 504 which will later be described.

On the other hand, if relative range mode is chosen, the system proceedsto step 503. Scaling the data in relative range is advantageous.Particularly, relative range mode minimizes the effect of aberrationsresulting from dirt or other imperfections on the substrate by ignoringa certain percentage of data points at the intensity extremes. Fromexperience, this range is typically from about 1% of the lowest valuesand 0.03% of the highest values. At step 503, the user enters the rangevalues or in the alternative, the system may provide for default values.For example, if the user enters 0.03% and 1% as the relative rangevalues and there are 100,000 pixels in the image, the brightest 30pixels (0.003×100,000) and dimmest 1000 (0.1×100,000) pixels are clipped(clamped to its next highest or lowest value respectively).

At step 504, the system determines if the dynamic range (i.e., the ratioof the count in the brightest pixel and the dimmest pixel) of the imageis high. If the ratio is high, the system prepares the data forlogarithmic scaling at step 505. Otherwise, the system proceeds to step506 where the data will be scaled linearly. In alternative embodiments,the system may prepare the data for square root scaling instead oflogarithmic scaling. In this manner, loss of valid data at the lowintensities having low photon counts are avoided, thereby, increasingthe resolution at these intensities.

At step 506, the system obtains the minimum and maximum photon countsfrom the data. The range between these two count values is used to scalethe data into digitized units. Preferably, the digitized units have arange from 0 to 255, each one representing a gray level or a color.Alternatively, other ranges such as from 0 to 63 may be used. Generally,the average photon count value is placed in the middle of scale.

Upon completion of the conversion process, an image file representingfluorescence intensity is created and stored in memory at step 507. Atstep 508, the system may optionally display the image file. Theintensity level of the displayed image varies from region to regionaccording to the binding affinity of the targets to the polymer sequencetherein. The brightest signals typically represent the greatest bindingaffinity while signals of lesser intensity represent lesser degrees ofbinding affinity.

As described, data are collected over regions substantially smaller thanthe area in which a given polymer or feature is synthesized. Forexample, the length of a pixel is generally 1/4 to 1/10 the length of afeature (or the area of a pixel is 1/16 to 1/100 the area of a feature).Hence, within any given feature, a large number of fluorescence datapoints or pixels are collected.

A plot of the number of pixels versus the fluorescence intensity for ascan of a substrate synthesized with probes when it has been exposed to,for example, a labeled antibody will typically take the form of a bellcurve. However, spurious data are observed, particularly at higherintensities. Since it is preferable to used an average of thefluorescence intensity over a given synthesis region in determining therelative binding affinity, these spurious data points will tend toundesirably skew the data.

FIGS. 6a and 6b illustrate one embodiment of the of a system whichprovides for the removal of these undesirable spurious data points aswell as the determination of the relative binding efficiency of thesample from an average of the remaining data points.

Referring to FIGS. 6a and 6b, the system is initialized by requestingthe user to enter the name of a image file of interest. At step 601, thesystem retrieves the image file and displays the image on the imagedisplay. The system then prompts the user to select the area of theimage to analyze (i.e., clicking the mouse on each of the four cornersof the synthesis area at step 602. Next, at steps 603 and 604, thesystem prompts the user for the number of cells located horizontally andvertically on the substrate. From the information entered by the userand the image file, the system creates a computer representation of ahistogram or each cell at step 605. The histogram (at least in the formof a computer file) plots the number of pixels versus intensity.

At step 606, the main data analysis loop is performed for each synthesissite. Analyzing the histogram for the respective synthesis site, thesystem calculates the total intensity level and number of pixels for thebandwidth centered around varying intensity levels. For example, asshown in the plots to the right of step 606, the system calculates thenumber of pixels in the bandwidth using boxcar averaging technique. Thisprocess is then repeated until the entire range of intensities have beenscanned. At step 607, the system determines which band has the highesttotal number of pixels. The data from this band is used to derivestatistical data for each synthesis site. The statistical data includethe peak value, mean intensity and standard deviation of intensitylevel. Thus, data that are beyond this band are excluded from thestatistical analysis. Assuming the bandwidth is selected to bereasonably small, this procedure will have the effect of eliminatingspurious data located at both the higher and lower intensity levels.This loop is repeated until all the cells have been processed.

At step 610, an image in the form of a grid representing the substrateis displayed. Each block in the grid represents a region synthesizedwith a polymer sequence. The image intensity of each region will varyaccording to the binding affinity between the polymer sequence andtargets therein. Statistical data, such as the peak and averageintensity corresponding to each region are also displayed.

To improve imaging, pixels located at transitions between synthesisregions are ignored. The image, in some instances, requires only onepixel space between the cells when the transition of the intensitybetween the synthesis regions is sharp and distinct. However, if thetransition is fuzzy or smeared, the user, at step 611, can select agreater number of pixel spaces between the cells to increase imageresolution. If the user enters a value indicating a greater number ofpixel spaces is desired, the system at step 616 reformats the imageaccordingly.

At step 612, the system retrieves the file created during the synthesisprocess of the substrate being analyzed. The synthesis file containssequence information as a function of location. The system integratesthe synthesis file with the image file and sorts the data therein.Through this process, the molecular sequence of complementary probes andthe intensity as a function of location is available.

Further, the user, at step 614, may analyze a specific synthesis regionwithin the grid. If instructed, the system will display thecorresponding substrate position, number of photons, number of pixelsand the molecular sequence at that synthesis site. The data analysissoftware also provides the user with many functions which are common toimage processing, such as magnification and image enhancement.

V. Conclusion:

The present invention provides greatly improved methods and apparatusfor detection of intensity of fluorescence on a substrate. It is to beunderstood that the above description is intended to be illustrative andnot restrictive. Many embodiments will be apparent to those skilled inthe art upon reviewing the above description. Although the detectionapparatus has been illustrated primarily herein with regard to thedetection of marked targets, it will readily find application in otherareas. For example, the detection apparatus disclosed herein could beused in the fields of catalysis, DNA or protein gel scanning, and thelike. The scope of the invention should, therefore, be determined notwith the reference to the above description, but should instead bedetermined with reference to the appended claims, along with the fullscope of equivalents to which such claims are entitled.

What is claimed is:
 1. An apparatus for detecting fluorescently markedregions on a first surface of a substrate, said apparatus comprising:anexcitation light source; an optical train for directing an excitationlight from said excitation light source at said substrate, andseparating reflected excitation light from said first surface fromfluoresced light from said first surface, said optical train comprisinga spatial filter having a first lens and a second lens and a confocalpinhole located between said first lens and said second lens, a beamsplitter cube, and a dichroic mirror for passing light having awavelength of about said fluoresced light and reflecting light having awavelength of about said excitation light, an optical lens and amicroscope objective for directing said light at said substrate; afocusing system for determining a focal plane of said excitation lightpassing through said optical train, said focusing means providing datafor locating said first surface at said focal plane; a detector fordetecting said fluoresced light from said fluorescently marked regionsin response to said light; an x-y-z translation system for translatingsaid substrate from a first position to a second position; a flow cellmounted on said translation system, said flow cell comprising a mountingsurface with a cavity therein, said mounting surface including a meansfor mounting said substrate thereon, and maintaining a sealedrelationship with said substrate, whereby said first surface of saidsubstrate is in fluid communication with said cavity, said cavity havingan inlet and an outlet, and said inlet being connected to a pump fortransferring materials into said cavity through said inlet and out ofsaid cavity through said outlet; and a storage system for storing a setof values representing an intensity of said fluoresced light as afunction of the location on said substrate fluorescing said fluorescedlight.
 2. The apparatus as recited in claim 1, further comprising avideo display for displaying said values representing the intensity ofsaid fluoresced light as a function of location on said substrate. 3.The apparatus as recited in claim 1, wherein said focusing meanscomprises:a photodiode for generating a voltage representing anintensity of said light reflected from said substrate; a focusing lensfor focusing said reflected excitation light from said substrate, fromsaid optical train at said photodiode; and a means for moving saidsubstrate relative to a microscope objective until said intensity ofsaid reflected excitation light focused at said photodiode from saidsubstrate substantially reaches a maximum.
 4. The apparatus as recitedin claim 3, wherein a confocal pinhole is located between said focusinglens and said photodiode.
 5. An apparatus as recited in claim 1, whereinsaid detecting means comprises:a photomultiplier tube; and a lens forfocusing said fluoresced light separated by said optical train, at saidphotomultiplier tube.
 6. An apparatus as recited in claim 5, wherein aconfocal pinhole is located between said focusing lens and saidphotomultiplier tube.
 7. An apparatus as recited in claim 5, whereinsaid photomultiplier tube is coupled to a means for collecting pulsesgenerated by said photomultiplier tube in response to said fluorescedlight, said means for collecting pulses being connected to aprogrammable computer for storing and analyzing said pulses.
 8. Anapparatus as recited in claim 1, further comprising means forcontrolling temperature in said flow cell, said means for controllingtemperature including a recirculating bath device for circulating waterthrough channels disposed in said flow cell.
 9. A method for detectingfluorescently marked regions on a substrate, said method comprising thesteps of:immobilizing said substrate on a body, said body comprising amounting surface having a cavity disposed therein, said substrate beingimmobilized on said mounting surface such that a probe array fabricatedon said substrate is in fluid communication with said cavity, saidcavity comprising a inlet and a outlet for flowing fluids into andthrough said cavity; directing an excitation light from an excitationlight source at said substrate; auto-focusing said substrate in a focalplane of said excitation light; exciting a first region of saidsubstrate with said excitation light from said excitation light source,said excitation light source having a first wavelength; detectingfluoresced light from said substrate in response to said excitationlight, said fluoresced light having a second wavelength, said detectingcomprising separating said light having a first wavelength from saidlight having a second wavelength and detecting said light having asecond wavelength; exciting a subsequent region on said substrate;repeating steps of detecting and exciting a subsequent region until allregions of said substrate have been excited; and processing and storingsaid fluoresced light to generate a 2-dimensional image of saidsubstrate.
 10. The method as recited in claim 9 wherein said bodyfurther comprises a temperature controller for controlling thetemperature in said cavity.
 11. The method as recited in claim 9,wherein said step of detecting comprises the steps of:collecting saidfluoresced light through optics; and directing said fluoresced lightfrom said optics onto a detector.
 12. The method as recited in claim 9,wherein said step of exciting said subsequent region comprises the stepof translating said substrate to allow said excitation light to excitesaid subsequent region.
 13. The method as recited in claim 9, whereinsaid auto-focusing step comprises the steps of:a) focusing a firstsurface of said substrate; b) focusing a second surface of saidsubstrate; and c) finely focusing said second surface.
 14. The method asrecited in claim 13, wherein said step of focusing said first surfacecomprises the steps of:directing said excitation light at saidsubstrate, said excitation light being reflected by said substrate;focusing said reflected excitation light from said substrate through aconfocal pinhole; detecting an amount of said reflected excitation lightpassing through said confocal pinhole, said confocal pinhole configuredsuch that said amount of said reflected excitation light is atsubstantially a maximum when said first surface is located insubstantially a focal plane of said excitation light; moving saidsubstrate closer to said excitation light and repeating the directing,focusing, detecting and moving steps until said amount of reflectedexcitation light passing through said confocal pinhole and detected insaid detecting step has peaked.
 15. The method as recited in claim 14,wherein said step of focusing said second surface comprises the stepsof:after focusing said first surface, first moving said substrate closerrelative to said excitation light, a distance which said substrate ismoved being equal to about half a thickness of said substrate; directingsaid excitation light at said substrate, said excitation light beingreflected by said substrate; focusing said reflected excitation lightthrough said confocal pinhole; detecting said amount of reflectedexcitation light passing through said confocal pinhole; second movingsaid substrate closer relative to said excitation light and repeatingthe second moving, directing, focusing, and detecting steps until saidamount of reflected excitation light passing through said confocalpinhole and detected in said detecting step has peaked.
 16. The methodas recited in claim 15, wherein said step of finely focusing said secondsurface comprises the steps of:directing said excitation light at saidsubstrate; focusing said reflected excitation light through saidconfocal pinhole; detecting said amount of reflected excitation lightpassing through said confocal pinhole; and moving said substrate fartherrelative to said excitation light and repeating the directing, focusing,detecting, determining, and moving steps until said amount of reflectedexcitation light passing through said confocal pinhole has reached adesired value.
 17. The method as recited in claim 13, wherein said stepsa-c are repeated for each corner of said substrate.
 18. The method asrecited in claim 17, further comprising the step of interpolating afocus position of each corner to determine said focus position of eachregion of said substrate.